CHAPTER - 2
INTRODUCTION TO
PEROXIDASES, GLUCOSE OXIDASE,
URICASES AND CATALASES
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
31
SECTION 2.1: PEROXIDASE
2.1.1 Introduction
Peroxidases (donor: H2O2 oxidoreductase, POD; EC 1.11.1.7) are a class of
enzymes extensively distributed among higher plants, animals and microorganisms
[1]. Most of the peroxidases are heme proteins and contain ferriprotoporphyrin IX as
the prosthetic group having the molecular weight ranging from 30,000 to 150,000 Da.
They catalyze the reduction of peroxides (H2O2) but also oxidize a variety of organic
and inorganic compounds. Peroxidases are involved in balancing and controlling the
biosynthesis of plant growth hormone, serving as a blanching indicator due to its
thermal stability under limited heat treatment, and is widely employed in
microanalysis due to its ability to yield chromogenic products at low concentrations
with relatively good stability [2-4].
Peroxidases have acclaimed a prominent position in biotechnology and
associated research areas and they are one of the most extensively studied groups of
enzymes. Commercially available peroxidase is widely employed for removal of
phenols and amines from industrial wastewater, bleaching of industrial dyestuff,
lignin degradation, fuel and chemical production from wood pulp and in various
organic syntheses [5].
2.1.2 Different classes of peroxidase
The term peroxidase represents a group of specific enzymes, such as NADH
peroxidase (EC 1.11.1.1), glutathione peroxidase (EC 1.11.1.9), and iodide
peroxidase (EC 1.11.1.8), as well as a group of nonspecific enzymes that are known
as peroxidases. Two super families of heme peroxidases have been identified [6]; one
isolated from plants, fungi, and bacteria and another from mammals. The
homologous enzymes of the plant super family have been sub-divided into three
classes [7]
• Class I - Peroxidases of prokaryotic origin which are intracellular
• Class II - Secretory fungal peroxidases and
• Class III - Extra cellular plant enzymes secreted into the cell wall or the
surrounding medium.
2.1.3 Structure of horseradish peroxidase (HRP)
All plant peroxidase enzymes share the same general structure, consisting of
ferriprotoporphyrin IX as a prosthetic group and ten α-helices. The class III
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
32
peroxidases also contain three extra α-helices, besides a few highly conserved amino
acids and four disulfide bridges [8]. Figure 2.1.1 shows the three dimensional
representation of the X-ray crystal structure of HRP isoenzyme C. The heme group
(colored in red) is located between the distal and proximal domains in which each
contains one calcium atom (shown as blue spheres). α-Helical and β-sheet regions of
the enzyme are shown in purple and yellow, respectively.
Figure 2.1.1. Structure of Horseradish Peroxidase (HRP)
2.1.4 Probable reaction mechanisms of catalytic activity of peroxidase
Peroxidase catalyzes the oxidation of a wide variety of substrates, using H2O2
or other peroxides as the primary substrate. Catalysis of peroxidase is associated with
four types of activity namely, Peroxidic, Oxidative, Catalytic and Hydroxylation
reaction [1]. The plant peroxidase protein sequence is characterized by the presence of
highly conserved amino acids, such as two histidine residues interacting with the
heme (distal and proximal histidines) and eight cysteine residues forming disulfide
bridges. The distal histidine is necessary for the catalytic activity. These histidine
residues are present in all known heme-containing peroxidase sequences [9].
2.1.4.1 Peroxidic reaction
It is the reaction involving peroxidase and H2O2 (or any peroxide) resulting in
the formation of oxygen free radicals or hydroxyl radicals. The generated radicals
react with other activated aromatic co-substrates to form the respective products [10].
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
33
2.1.4.2 Oxidative coupling reaction
It involves trapping of free radical species of one co-substrate to get oxidized
to electrophillic species, which couples with the coupling agents of other co-substrates
[11] forming an intense colored product that could be characterized by spectroscopic
instruments.
2.1.4.3 Catalytic reaction
It provides an alternative reaction pathway and stabilizes the intermediates by
reducing the transition energy of the reaction, thereby increasing the number of
reactant molecules with sufficient energy to reach the activation energy and to form
the product [12].
2.1.4.4 Hydroxylation reaction
Hydroxylations belong to the oxygen transfer reactions introducing the
hydroxyl group (.OH) into organic molecules, primarily via the substitution of
functional groups or hydrogen atoms [13].
In general, as a whole, catalytic cycle of peroxidase involves distinct
intermediate enzyme forms. In the catalytic cycle of heme peroxidases, reaction of the
Fe(III) “resting state” active site with peroxide produces an unstable intermediate
called compound I that contains Fe(IV)=O porphyrin π-cation radical and a cation
radical with the consequent reduction of peroxide to water, wherein a distal Histidine
residue plays an essential role in the initial two electron oxidation of resting state
enzyme. A distal Arg residue assists in this process [14, 15].
Then, compound I oxidizes an electron donor substrate to give compound II
(same oxyferryl structure, but protonated), releasing a free radical. Compound II is
further reduced by a second substrate molecule regenerating the iron (III) state and
producing another free radical [16]. Figure 2.1.2 shows the peroxidative and
hydroxylic cycle of peroxidase.
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
34
Figure 2.1.2. Peroxidase reaction cycles.
The hydroxylic cycle (represented with orange arrows) can generate ROS such
as .OH and HOO. by two different routes (i, ii) (Figure 2.1.2). The source of e- and H+
(1) can be either auxin or other reducing molecules. The peroxidative cycle
(represented in green) can oxidize various substrates, represented by XH and X. for
the reduced and oxidized forms, respectively. X. has three major fates: auxin
catabolism (*), cell wall component polymerization (**) and NAD(P)H oxidation
(***) via a non-catalytic reaction (2) (Figure 2.1.2). NAD(P)H oxidation produces
superoxide, which is immediately converted either spontaneously or by superoxide
dismutase to H2O2 and O2 (3) (Figure 2.1.2). Hydroxylic and peroxidative cycles can
both regulate the H2O2 level. The O2.- released during the oxidative cycle by NADPH
oxidase can convert peroxidase into compound III, which catalyzes the generation of .OH from H2O2 in the cell wall.
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
35
2.1.5 Physiological relevance of peroxidase
Peroxidases are involved in a wide range of physiological processes related to
plant growth and development [17]. They play an important role in the plant defense
system mechanism by the oxidation of endogeneous phenolic compounds to quinones,
which are toxic to the invading pathogenic organisms and pests [18]. It can also
oxidize the growth hormone auxin, as well as other substrates producing H2O2 and
hydroxyl radicals, which are involved in oxidative burst and cell elongation. By
generating hydroxyl radicals (.OH), peroxidases play a crucial role in seed protection
as well as in the initial days of germination by reducing pathogenic attack. The
diversity of the reactions catalyzed by plant peroxidases explains the implication of
heme proteins in a broad range of physiological processes, such as auxin metabolism,
lignin and suberin formation, cross linking of cell wall components, defense against
pathogens [9], and HRP isoenzymes including indole-3-acetic acid (IAA) metabolism
[19]. It often contributes to deteriorative changes in flavor, color, texture, and
mouthfeel in raw and processed fruits and vegetables [3].
2.1.6 Application of peroxidase
In analytical applications the enzyme must be present in saturated amounts, to
make sure that the H2O2 produced in the test is stoichiometrically converted into a
colored product [20]. Some of the most important applications of peroxidase which
are being used have been elaborated in Table 2.1.1.
2.1.6.1 Peroxidase in immunoassay
Enzyme immunoassay or ELISA test, is the most common technique used for
labeling an antibody are simple and reliable way of detecting toxins, pathogens,
cancer risk in bladder and many other analytes [21]. ELISA tests have been developed
for screening monoclonal antibodies against mycotoxins [22], cystic fibrosis mutation
in blood [23], human tumour necrosis factor alpha, using biospecific antibodies [24].
2.1.6.2 Peroxidase in medical diagnostic kits
Peroxidase has been used for analytical applications in diagnostic kits for the
quantification of important biomolecules such as uric acid [25], glucose [26],
cholesterol [27], nucleic acids [28] etc by using their respective oxidase enzymes.
2.1.6.3 Peroxidase as bioremediation of waste water
Lignin peroxidase (LiP) and manganese peroxidase (MnP) may be
successfully used for bio-pulping and bio-bleaching in the paper industry. HRP
catalyzes the oxidation of aqueous phenols by H2O2 to produce free radicals that
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
36
spontaneously interact to form polymers and oligomers of high molecular weight and
water-insoluble precipitates. These products are precipitated from the solution and can
be removed from water or wastewater by filtration or sedimentation [29].
2.1.6.4 Peroxidase as biosensors
Biosensor has attracted considerable attention as the potential successor to a
wide range of analytical techniques owing to its unique characteristic of specificity.
Enzymes immobilized on the electrode surface get oxidized by H2O2 and then reduced
by electrons provided by the electrode. During the electron transfer, electrons act as
second substrate for the enzymatic reaction, resulting in the shift of electrode
potential, with measured current being proportional to the H2O2 concentration [30].
Figure 2.1.3 shows the schematic representation of a biosensor. Peroxidase-based
biosensors have been used for the determination of alcohols, glutamate and choline
[16].
Figure 2.1.3 A schematic representation of a biosensor with electrochemical transducer.
2.1.6.5 Peroxidase in agricultural technologies
Peroxidase has a potential for soil detoxification, for instance, herbicides such
as atrazine and triazine gets bio-transformed to the less toxic compounds by P.
chrysosporium which contains LiP and MnP [31], [32]. The use of peroxidase to
improve dewatering of slimes and for the polymerization of humic acid in soil organic
matter has also been reported. Chlorinated phenols and anilines get transformed in
soil by oxidative and detoxification coupling reactions mediated by laccase,
peroxidase or metal oxides as birnessite [33].
2.1.6.6 Peroxidase in pharmaceuticals
HRP/IAA represents an efficient system for enzyme/prodrug-based anticancer
approach [34]. Another application of plant peroxidases in the field of organic and
polymer synthesis is related to the coupling of catharanthine and vindoline to yield α-
3′,4′-anhydrovinblastine which is a part of most of the curative regimes used in cancer
chemotherapy [35].
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
37
Table 2.1.1 Peroxidase based analytical methods
Sl.
No.
Analytical Method Reagent co-substrate Analyte determined Reference
01
Spec
trop
hoto
met
er
MBTH-DMAB/HRP/GOD/α-glucosidase H2O2, glucose & maltose [11]
ABTS POD activity-broccoli processing wastes [36]
TMB POD activity in soil [37]
TMPD Inactivation of prostaglandin-H-Synthase [38]
Guaiacol POD activity - plant samples [39]
Benzidine/ p-phenylene diamine POD activity in mitochondrial membrane [40]
o-phenylenediamine (OPD) HRP [41]
Eriochrome Blue Black R (EBBR) Degradation of EBBR, fluorescein [42]
o-dianisidine, TMB & OPD POD mechanism [43]
Alizarin/ H2O2/HCl Alizarin degradation [44]
Phenol/4-AAP O2- and SOD activity in vegetables [45]
MBTH/DBZ H2O2 and glucose [46],
2,4-DMA; PPDD-DMAB; PPDD-NEDA HRP and H2O2 [47-49]
Pyrogallol/Veratryl alcohol Effect of veratryl alcohol on LiP [50]
Catechol, o-dianisidine, OPD POD activity [51]
Phenol-4-sulphonic acid & 4-AAP H2O2 and HRP activity [52]
Pyrocatechol/aniline HRP activity [53]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
38
02 ELISA Dopamine Dopamine in serum [54]
03 Chemiluminescence-
CL
Luminol HRP [55]
4-(1,2,4-triazol-1-yl)phenol/luminol H2O2 in rain water [56]
04 Flow injection
analysis
3-Aminophthalic acid/Silane; Phenol/AAP Glucose [57, 58]
Luminol, p-iodophenol Sulfamethoxypyridazine in milk sample [59]
05 GC-MS Allergenic eugenol Eugenol removal from rose essential oil [60]
06 Potentiometric 4-fluorophenol H2O2 [61]
07 Photo electrochemical
immunoassay
CdS quantum dots/photoactive antibody-
antigen
Mouse IgG (antigen, Ag) [62]
08
Am
pero
met
ric
Amine group containing polymers H2O2 quantification [63], [64]
Cyanuric chloride [65]
Halloysite & chitosan nano composite H2O2 in milk samples [66]
Dichlofenthion/Organophosphorus hydrolase Dichlorofenthion, pesticides [67]
Polyacrylamide microgels Acetaminophen [68]
Poly(2,5-DMA) Glucose [69]
09 Electro chemical
immunoassay
p-aminophenol Cucumber mosaic virus [70]
3-Hydroxyl-2-amino pyridine α-Fetoprotein [71]
4-chloro-1-naphthol Cancer biomarker-Prostate antigen [72]
o-tilidine Carcinoembryonic Antigen [73]
10 Microtiter plate reader Al(III)Octaethylporphyrin/4-fluoro phenol Glucose [74]
11 Chronometric Ascorbic acid/Benzidine HRP and tea enzyme [75]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
39
12 Resonance scattering
assay
Tetradecyldimethylbenzylammonium
chloride
Glucose [76]
13 Capillary electrophoresis Phenol/4AAP/Amberlite IRA-743 resin H2O2 in honey and minerals [77]
14 LC/MS Bisphenol A (BPA) BPA removal from water, waste water [78]
15
Spec
trof
luor
imet
ry
3-(4-hydroxyphenyl) propionic acid H2O2 and Glucose [79]
Amplex red; Fluorescent gold nanocluster H2O2 [80], [81]
Sesamol (3,4-methylenedioxy phenol) Thyroid stimulating hormone [82]
Tyrosine HRP activity [83]
Dihydroxyphenoxazine HRP and H2O2 [84]
Homovanillic acid H2O2 in honey samples [85]
1-Hydroxypyrene Ozone in the air [86]
Hydroxynaphthaldehyde thiosemicarbazon H2O2/-O-O-H bond in PEG [87]
16 ECIS Polymyxin H2O2 and O2 reduction, biofeul cells [88]
Polyacrylamide/SWCNT H2O2 [89]
(CaCO3–AuNPs) inorganic composite [90]
17 Voltammetry-ELISA Alkaline phosphatase/3-indoxyl phosphate Pneumolysin-toxin respiratory to infection [91]
3,3′-diaminobenzidine CEA [92]
18 Batch process Diethyl aminoethyl cellulose
α-naphthol removal in waste water [93]
19 UV irradiation Semiconducting iron-doped titanate Peroxidase activity of HRP
[94]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
40
20
Cyc
lic v
olta
mm
eter
Polyaniline/MWCNT
H2O2
[95]
4-ethylnylphenyl [96]
(3-aminopropyl)trimethoxy silane/1,4-
benzoquinone titanate nano tube
[97]
Au NPs –thionine/chitosan [98]
Poly(3,4-ethylenedioxythiophene) - Poly(
styrene sulfonic acid)/Au nanocomposite
[99]
Bismuth oxide (Bi2O3) NPs/ MWCNT [100]
Anthraquinone 2-carboxylic acid [101]
Hydroquinone/Nanoclay Glyphosate [102]
Poly(diallyldimethylammonium chloride) NO2• radicals induced DNA damage [103]
21 Conductivity cell Polyethylene terephthalate/ABTS HRP in Sub femtoliter volume [104]
22 Coulometric biosensor 1,4-hydroquinone/γ-aminopropyl-
diethoxymethyl-silane
H2O2 molecules present in cells [105]
Note: 1. ECIS: Electrochemical Impedance Spectroscopy; CV: Cyclic Voltammeter; MWCNT: Multi walled carbon nanotubes;
SWCNT: Single walled carbon nanotubes; NPs: Nano particles.
2. ABTS: 2,2-Azinobis(3-ethylbenzthiazoline-6-sulfonicacid; MBTH: 3-methyl-2-benzothiazolinone hydrazone hydrochloride; DMAB:
Dimethylamino benzoic acid; TMB: 3,3′,5,5′- Tetramethylbenzidine; TMPD: tetramethyl-p-phenylenediamine; DBZ: 10,11-dihydro-5H-
benz(b,f)azepine); 2,4-DMA: 2,4-Dimethoxyaniline; PPDD: Paraphenylenediamine dihydrochloride; NEDA: N-(1-naphthyl)
ethylenediamine dihydrochloride; 4-AAP: 4-aminoantipyrine; POD: Peroxidase; HRP: Horseradish peroxidase; CEA: Carcinoembryonic
Antigen; LiP: Lignin peroxidase; PEG: Polyethylene glycol.
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
41
SECTION 2.2: GLUCOSE OXIDASE
2.2.1 Introduction
Glucose oxidase (GOD) (β-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4)
is a flavin containing glycoprotein with high-mannose type carbohydrate content of
10-16% of its molecular weight. The carbohydrate moieties are either N or O- which
are glycosidically linked to the protein. The enzyme activity was first reported by
Muller (1928) [106] in extracts of Aspergillus niger and subsequently, this enzyme
was purified. Müller in 1928 established that the enzyme catalyzes the oxidation of
glucose to gluconic acid in the presence of dissolved oxygen. The fungal enzyme is a
homo dimer made up of two identical subunits each having molecular weight of
approximately 80,000 Daltons [107]. Each monomer contains a non-covalently bound
FAD molecule, which is acting as a redox carrier in catalysis [108]. Sources of GOD
include extracts from Aspergillus niger, Penicillium amagasakiense, Penicillium
vitale and Penicillium glaucum. Isolation of GOD from a number of sources has been
reported and these include red algae, citrus fruits, insects, bacteria and moulds [109].
GOD is produced industrially as a by-product of the gluconic acid fermentation from
A. niger, P. amagasakiense and P. vitale [110].
GOD is the most widely employed enzyme as an analytical reagent and
especially it is useful for the selective determination of glucose, which is an analyte of
clinical as well as of industrial importance. The relatively low cost and good stability
make the glucose/GOD system, a very convenient model for method development
particularly in the area of biosensors.
2.2.2 Structure of Glucose oxidase
GOD is composed of two polypeptide chains of approximately equal
molecular weight held together by disulfide bonds with carbohydrate content
amounting to 16 %. The primary structure of GOD from Aspergillus niger has single
polypeptide chain of one subunit of 583 amino acid residues [111]. The crystal
structure of the enzyme was solved at 2.3 A° resolutions. The protein is glycosylated,
containing between 11 and 30 % carbohydrates: mostly amino- and neutral-sugars
[112], [113]. Dissociation of subunits is possible only under denaturing conditions
and is accompanied by the loss of coenzyme FAD [114]. The structure of GOD is
shown in Figure 2.2.1.
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
42
Figure 2.2.1. Structure of Glucose oxidase from Aspergillus Niger
2.2.3 General reaction mechanism of Glucose oxidase
GOD catalyzes the oxidation of β-D-glucose using molecular oxygen as an
electron acceptor to D-glucono-δ-lactone, which subsequently gets hydrolyzed
spontaneously to gluconic acid and H2O2 [115], and the reaction involves two steps
one reductive and another oxidative as shown in Scheme 2.2.1.
In the reductive half reaction, GOD catalyzes the oxidation of β-D-glucose to
D-glucono-δ-lactone, which is non-enzymatically hydrolyzed to gluconic acid.
Subsequently the FAD ring of GOD gets reduced to FADH2 [116]. In the oxidative
half reaction, the reduced GOD is reoxidized by oxygen to yield H2O2. The H2O2 is
cleaved by catalase to produce water and oxygen. Witteveen et al., (1992) found that
the enzyme lactonase (EC 3.1.1.17) in A. niger to be responsible for catalyzing the
hydrolysis of glucono-δ-lactone to gluconic acid [117].
Scheme 2.2.1 General reaction mechanism of GOD in presence of glucose as a
substrate
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
43
2.2.3.a The probable reaction pathway occurring in the colorimetric method
Enzymatic method of determination of blood glucose level using a
chromogenic substrate involves the following mechanism of generation of the colored
reaction product for spectrophotometric method.
2.2.3.b The probable reaction pathway in the electrochemical method
The electrocatalytic processes that occur at the electrode surface can be
depicted as follows
2.2.4 Physiological relevance of Glucose oxidase
GOD in culture filtrates of Talaromyces flavus is responsible for inhibition of
germination of microsclerotia of V. dahliae, a plant pathogen and it is a major factor
in biocontrol of V. dahliae by T. flavus [118]. The inhibitory effect of GOD activity
on germination and melanin formation secreted by T. flavus, retards hyphal growth
and kills microsclerotia of V. dahliae and Sclerotium rolfsii in vitro, probably by
generating toxic peroxide in soil [119, 120]. It is also involved in the biocontrol of
Verticillium wilt of eggplant by T. flavus [121].
GOD gene expression of T. flavus in cotton and tobacco reduces fungal
infection by generating H2O2, in the presence of glucose, which is toxic to
phytopathogenic fungi responsible for economically important diseases in many crops
[122]. It is also involved in disease resistance conferred by gene expression for the
transgenic potato plants [123]. Use of GOD show that prolonged exposure to
moderate concentrations of H2O2 decreases insulin receptor signaling for cellular
mechanism in 3T3-L1 Adipocytes [124]. It is also used as antibacterial in
combination with antibiotics for ocular pathogens [125].
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
44
2.2.5 Applications of Glucose oxidase
GOD has gained considerable commercial importance during the last few
years due to its multitude of applications in chemical, pharmaceutical, clinical,
biotechnology and other industries. Some of its important current applications in
industry have been reviewed below.
2.2.5.1 Biofuel cells
Biofuel cells consist of two electrode set modified by biocatalytic enzymes to
specifically oxidize/reduce substrate cells by converting biochemical energy into
electrical energy. One approach towards the design of an implantable, membraneless
and biocompatible biofuel cell consists of catalyzing the oxidation of glucose at the
anode using either GOD or glucose dehydrogenase enzyme. These enzymes are
coupled to the reduction of dioxygen at the cathode by a dioxygen-reducing enzyme
such as laccase, bilirubin oxidase etc [126, 127].
2.2.5.2 GOD based biosensors for glucose assessment
GOD has been employed as the biological recognition component in a wide
variety of biosensors for glucose coupled with peroxidase. Some biosensors use
sensitive fluorescence measurements, monitoring changes in the intrinsic FAD
fluorescence of GOD [109]. Some of the recent GOD based biosensors have been
listed in Table 2.2.1.
2.2.5.3 Food and beverage additive
In order to prolong shelf life of foods and beverages, GOD/CAT is used to
remove glucose during the manufacture of egg powder, preventing browning for use
in baking industry, in bread and croissants [128]. This enzyme system is shown to
control non-enzymatic browning during fruit processing and puree storage. The
addition of GOD leads to an increase in the elastic and viscous moduli of wheat and
rice flour dough [129]. GOD is also used to prevent color and flavor loss as well as to
stabilize color and flavor in beer, fish, tinned foods and soft drinks by removing
oxygen from foods and beverages [130, 131].
2.2.5.4 Textile industry
GOD has application in the textile industry as a method for producing H2O2
for bleaching and immobilization of GOD covalently on alumina and glass supports
results in higher recoveries [132]. The H2O2 produced when tested for bleaching
scoured woven cotton fabric was found comparable to standard bleaching processes.
No stabilizers were needed since the gluconic acid produced itself acted as a
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
45
stabilizing agent [133]. GOD is used along with POD in dyeing baths for decoloration
process and bleaching of natural fibers in textile industries [134].
2.2.5.5 GOD as antagonist in various medical fields
GOD is reported to have the best antagonistic effect against different food-
borne pathogens such as Salmonella infantis, Clostridium perfringens and others
[135]. It has been used as an ingredient of toothpaste [136], and in food preservation
[137]. GOD has been proposed as an anticancer drug because of the damage caused to
cancerous tissue and cells as a result of H2O2 formation [138]. GOD has also been
used as anti-microbial agents in oral care products [139]. The H2O2 produced by GOD
acts as a bactericide and has the ability to kill Streptococcus mutans which appears to
be enhanced by the fusion of enzyme with heavy chain antibodies [140].
2.2.5.6 Gluconic acid production and application
GOD is also used as a commercial source of gluconic acid, which can be
produced by the hydrolysis of δ-glucono-1,5-lactone, the end product of GOD
catalysis. Gluconic acid has been used as a food additive to act as an acidity regulator,
in sterilization solution or bleaching in food manufacturing and as a salt in chemical
components for medication. It also used as a mild acidulant in metal and leather
industries. It has even been used in the construction industry as an additive to cement
in order to increase its resistance and stability under extreme weather conditions.
Gluconic acid occurs naturally in honey, fruits and wine [141], [142].
2.2.5.7 GOD in wine industry
GOD has potential for use in wine industry to lower the alcohol content of
wine through the removal of some portion of the glucose before converting to alcohol.
An alternative approach was introduced with the concept of treating grape juice from
mature fruits with GOD to reduce the glucose content (up to 50 % of grape sugar) of
the juice, which after fermentation produces wine with reduced alcohol content [143].
GOD was able to inhibit spoilage of wine through its bactericidal effect on acetic acid
bacteria and lactic acid bacteria during the fermentation process which insists lesser
quantity of preservatives are needed to be added to the wine [144].
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
46
Table 2.2.1. Glucose oxidase based analytical methods
Sl. No. Analytical Method Reagent co-substrate Analyte determined Reference
01 Spectrophotometer
Au NPs/N-methylphenazonium methyl
sulphate/2,6-dichloroindophenol
Efficiency of Au NP for enzymatic activity of
GOD
[145]
Triphenylmethane dyes/Benzoquinone GOD as bioanodes in biofuel cells [146]
Poly(Styrene-glycidylmethacrylate Kinetic parameters for GOD enzyme [147]
Polystyrene/AuNPs/dendritic surfactant Nano/microstructures on activity of GOD [148]
02 Chemometric - Characteristics of wheat bread dough [149]
03 Hronopotentiometric Polyaniline/Glutaraldehyde/Graphite Electrochemical detection of glucose [150]
04 Flow injection
analysis
4-AAP/4-hydroxy benzoate Glucose in honey [151]
Chitosan–ferrocene/ Glutaraldehyde GOD and Gluconobacter oxydans biosensors [152]
05 Titrimetric - GOD [153]
06 Miniature chip-design GOD/bilirubin oxidase/NADPH Directed evolution of GOD/biofuel cells [154]
07
Am
pero
met
ric
Polyaniline/glutaraldehyde Glucose [155]
1, 1′-dimethylferricinium GOD activity of cultivation of A. niger [156]
Polypyrrole Characterization of GOD biosensor [157]
Poly-L-lysine/Nafion Glucose sensor and biofeul cells [158]
Graphene oxide and concanavalin A Activity of GOD [159]
Poly-m-phenylendiamine/Hexakinase In-vitro analysis of ATP [160]
08 Chemiluminescence
Bis(2,4,6-trichlorophenyl) oxalate/8-
anilinonaphthalene-1-sulfonic acid
17α-hydroxy progesterone and congenital
adrenal hyperplasia in neonates
[161]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
47
Luminol; Nafion/peroxyoxalate Glucose, uric acid; biosensor [162], [163]
09 Electro-
chemiluminescence
ZnO NPs/GOD/graphene/luminol Carcinoembryonic antigen-cancer biomarkers [164]
Poly(nickel(II)tetrasulfo phthalocyanine Glucose in serum samples [165]
poly(ethylenimine)/Luminol α-1-fetoprotein [166]
10 Chrono-coulometric IrO/Nafion/Ir sol/GOD/Au support Glucose biosensor [167]
11 Spectropolarimeter ZnO nano particles/GOD Photoelectrochemical - glucose biosensor [168]
12 ECIS/FTIR 1-butyl-3-methylimidazolium Glucose bioanode for biofuel cells [169]
13 Microchip &
Microarray technology
GOD/antibody/hexamethyl disiloxane
plasma -polymerized film
Development of protein chips for proteomics
of α-1-fetoprotein and β2-microglobulin
[170]
14
Cyc
lic
volta
mm
eter
Phenanthrenequinone tetrathiafulvalene Glucose/O2 –Biofuel cell [171]
DNA/Fe2+/CAT/Ru(bpy)3+3 in situ DNA damage [172]
N,N-diethylacrylamide/4-vinyl pyridine Electrochemical glucose biosensor [173]
1,1′-bis(4-carboxybenzyl)-4,4′-
bipyridiniumdibromide/TiO2/Viologen
Electrocatalytic activity of GOD for glucose
biosensor
[174]
Poly(allylaminehydrochloride) Glucose biosensor [175]
15 Electrochemical
immunosensor
p-benzoquinone/AuCl4− Detection of mouse IgG (antigens) [176]
Ferrocene/peptide wire/Ag nano rod α-tumor protein biomarker [177]
Colloidal Prussian blue/Au NPs/SPCE CEA/α-fetoprotein/Tumor markers [178]
16 Capillary
electrophoresis
1,4-benzoquinone/β-cyclodextrin Enzymolysis and enzyme inhibition assay [179]
17 Potentiometer GOD/ZnO/Ag wires Glucose micro sensor [180]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
48
18 Spectrofluorimeter Polypyrrole/FAD/GOD Stability of GOD [181]
Poly(vinyl alcohol)-pyrene/ GOD Glucose sensing [182]
Eu(III)-tetracycline GOD, H2O2 [183]
19 F- sensing microtiter
plate wells
Al(III)Octaethylporphyrin/
4-fluorophenol
Glucose in beverages [74]
20 Optical biosensor
arrays
silica sol–gel/tris(4,7-diphenyl-
1,10-phenanthroline)-Ru(dpp)3Cl2)
Glucose in beverages and blood samples [184]
21 ELISA/Capillary
immunosensor
PEG/POD-labeled antibody/antigen/
glucose/ascorbic acid/Amplex red
Human IgG/Drug screening and clinical
diagnostic application
[185]
22 Fluidic calorimeter Sb–Bi thin film as thermopile/GOD Glucose sensor [186]
23 Plasmon-resonance
sensors
Poly(dimethylsiloxane)/Au nano
spheres
Study of interaction of GOD with its natural
substrate glucose
[187]
24 Si - microcantilevers GOD Microcantilever /Glucose Glucose sensor [188]
25 Phosphorescence Mn-doped ZnS quantum dots/1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide
/N-hydroxy succinimide
Glucose in real serum samples [189]
26 ENDOR FAD in GOD G-Tensors of the FAD radicals in GOD [190]
Note: ECIS: Electrochemical Impedance Spectroscopy; ENDOR: Electron-Nuclear Double Resonance; SPCE: Screen printed carbon
electrode; bpy = 2,2′-bipyridine; ATP: Adenosine triphosphate FAD: Flavin adenine dinucleotide; PEG: Poly(ethyleneglycol)
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
49
SECTION 2.3: URICASE
2.3.1 Introduction
Uricase (UOx) (Urate oxidase, EC 1.7.3.3) is the copper-binding peroxisomal
liver enzyme, which catalyzes the oxidation of uric acid (UA), a product of purine
metabolism into a more water-soluble allantoin through a complex reaction
mechanism, and then it is freely excreted by kidneys along with urine. Hydroxyisouric
acid, the immediate product of UOx reaction is unstable and thus yields allantoin,
CO2 and H2O2, through nonenzymatic breakdown. UOx is utilized in biological
systems for the degradation of purines and it is present in both prokaryotes and
eukaryotes. This highly conserved endogenous enzyme is present in mammals but not
in humans [191], plants (soybean, wheat (Triticum aestivum), broad bean (Vicia faba)
[192], fungi (Candida) [193], yeasts and bacteria (Bacillus fastidiosus) [194]. Two
nonsense mutations found in human UOx gene, confirm at the molecular level, that
UOx gene in humans is non-functional. UA is a powerful scavenger of free radicals
and it has been proposed that it protects hominoids from oxidative damage and
prolongs life span [195].
UOx is used in humans for the control of increased serum UA in patients with
acute tumour lysis syndrome after receiving chemotherapy. Rasburicase (SR 29142),
a recombinant UOx expressed by Saccharomyces cerevisiae has been demonstrated to
be superior to allopurinol (uricostatic agent) in the control of UA [196]. However,
only few case reports address the potential role of UOx for treatment of severe
tophaceous gout in patient with end-stage renal disease and observed regression of
gout tophi [197]. The treatment was well tolerated in all patients and produced no
adverse effects [198].
2.3.2 Structure of Uricase
UOx is mainly localized in liver, where it forms a large electron-dense
paracrystalline core in many peroxisomes [199]. The enzyme exists as a tetramer of
identical subunits, each containing a possible type 2 copper-binding site [200]. UOx is
a homotetrameric enzyme containing four identical active sites situated at the
interfaces between its four subunits. UOx from A. flavus is made up of 301 residues
with molecular weight of 33438 Da. It is unique among the oxidases as it does not
require a metal atom or an organic co-factor for catalysis. Sequence analysis of
several organisms has shown that there are 24 amino acids which are conserved, and
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
50
of these, 15 are involved with the active site. The structure of UOx is depicted in
Figure 2.3.1.
Figure 2.3.1 Structure of Uricase enzyme
2.3.3 General reaction mechanism of Uricase
UOx catalyzes the in vivo oxidation of UA in the presence of O2 to produce
allantoin and CO2 as oxidation products of UA and H2O2 as a reduction product of O2.
Enzymes from bovine liver [201], bacteria and fungi [202] do not contain copper.
Soybean UOx is also devoid of copper, other transition metals, and common redox
cofactors, thus presenting a mechanistically intriguing problem of how triplet oxygen
is activated to react with the singlet urate molecule [203]. Although allantoin is the
ultimate product that is formed from the oxidation of urate, the evidences suggest that
urate is oxidized by the enzyme to a metastable compound which gets decomposed
into allantoin nonenzymatically. The other reaction products are H2O2 and CO2,
which are derived from C6 of urate [204]. However, the NMR data used to support
the assignment of 5-hydroxyisouric acid as the product of UOx reaction are not
sufficient to rule out other structures. Characterization of the reaction product by
using a full complement of specifically 13C-labeled urates augmented by isotope-
labeling studies has been done in H218O. With the aid of specifically labeled urates, it
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
51
was also possible to delineate the pathway for the conversion of the enzymatic
reaction product into allantoin [203].
The probable reaction pathways of UOx catalyzing UA occurring in some
colorimetric and electrochemical methods are as follows
2.3.3.a Colorimetric method
Enzymatic method of determination of blood UA using a chromogenic
substrate involves the following mechanism of generation of the colored reaction
product for spectrophotometric method
2.3.3.b Electrochemical method
The electrocatalytic processes that occur at the electrode surface can be
depicted as follows
2.3.4 Physiological relevance of Uricase
UOx belongs to purine degradation pathway and it prevents the accumulation
of UA in blood. The absence of UOx expression in human has both advantages and
disadvantages because UA is a potent antioxidant and it helps to reduce the presence
of free radicals in cells, but in high concentration it can accumulate in plasma and
induce high pathologies that may be fatal [205].
There are reports regarding the protection of neurological damage of cells by
the presence of UOx [206]. It is the absence of a functional UOx gene that
predisposes humans to hyperuricemia and gout. However, rather than being
advantageous, mutational loss of UOx causes fatal UA nephropathy in mouse [207].
Lack of UOx in humans results in plasma UA concentrations that are much higher
than in most mammals. When these concentrations exceed the solubility limit of about
7 mg/dL at physiological pH, UA may nucleate to form crystals in tissues and joints
leading to acute inflammatory response with acute pain [208].
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
52
Legumes: UOx is also an essential enzyme in the ureide pathway, where
nitrogen fixation occurs in the root nodules of legumes and is converted into
metabolites that are transported from the roots throughout the plant for amino acid
biosynthesis. In legumes, two forms of UOx are found: in roots, the tetrameric form;
and in the uninfected cells of root nodules, a monomeric form, which plays an
important role in nitrogen-fixation [209]. Increase in UOx activity in the presence of
thioredoxin appears to implicate a novel role for thioredoxin in the regulation of
enzyme activities involved in nodule development and nitrogen fixation [210].
Yeast UOx in plant hoppers: Microorganisms such as bacteria and yeasts
catalyze the oxidation of UA produced in purine breakdown to allantoin and
successively to allantoic acid, urea and ammonia. In symbionts UOx plays a key role
in the host’s utilization of stored UA which is essential for the host to grow normally
[211]. In addition, UOx activity was also detected in symbiotic insects, in brown plant
hoppers (Nilaparvata lugens) and the isolated symbionts, but not in aposymbiotic
insects [212].
2.3.5 Applications of Uricase
UOx has gained considerable commercial importance during the last few years
due to its multitude of applications in the chemical, medical, clinical chemistry,
biotechnology pharmaceutical and other industries. UOx has been the subject of much
research mainly due to its recent therapeutic and diagnostic applications.
2.3.5.1 PEG-uricase in the management of treatment-resistant gout and
hyperuricemia
UOx is formulated as a protein drug (rasburicase) for the treatment of acute
hyperuricemia in patients receiving chemotherapy. A PEGylated form of UOx
(poly(ethylene glycol) conjugates) is under clinical development for treatment of
chronic hyperuricemia in patients with "treatment-failure gout". UOx can be useful in
lowering side effect caused by chemotherapy which results from hyperuricemia
treatment as a consequence of tumour lysis [213]. UOx from A. flavus has been used
therapeutically for treating patients with hyperuricemia, including those treated with
cytoreductive drugs for a malignant hemopathy [214].
2.3.5.2 Uricase as biosensors
A brief introduction on principles of biosensors is provided under Section 2.1
of peroxidase chapter, sub section 2.1.6.4. Using the activity of UOx along with other
oxidoreductase enzymes usually peroxidase, many types of biosensors have been
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
53
developed and reported for the quantification of either UOx activity or H2O2/UA.
Some of the Uricase based methods reported recently in the literature have been listed
in Table 2.3.1.
2.3.5.3 Uricase in medical & clinical analysis
UOx has been used as a drug for rapid lowering of urate level which is needed
potentially in organ transplant patients with gout, tumor lysis syndrome, tophaceous
gout, renal failure, and acute gout attacks [215]. It has been used for the symptoms of
Lesch-Nyhan syndrome and confirmed the possible protection of neurological cells by
UOx enzyme [206]. It is also used as a peroxisomal marker [216] and is potentially a
good system for studying protein sorting into peroxisomes [200]. A recombinant
preparation of purified fungal (A. flavus) UOx (Uricozyme; Sanofi-Synthelabo)
enzyme is now available in the United States for this indication [217].
2.3.5.4 Uricase as polymer-conjugated therapeutic agent for immunological
studies
Polymer conjugation has so far been successfully used to enhance the
therapeutic potential of many pharmacologically active proteins and peptides since it
allows for the alteration of their physicochemical and biological properties improving
permanence in circulation, stability, solubility and reducing immunogenicity [218].
Poly(N-vinylpyrrolidone), poly(N-acryloilmorpholine) linear and branched PEG2, can
improve the immunogenic character of UOx. Immunological studies showed that
antigenicity and immunogenicity of UOx were altered by polymer conjugation to such
an extent that depended upon the polymer composition [213].
2.3.5.5 Uricase for color development in hair-dyeing and fur-dyeing
In usual hair-dyeing practice, the coloring reaction is initiated by mixing H2O2
directly with the organic reactants and such direct use of H2O2 at relatively high
concentrations may damage hair and skin due to the strong oxidizing power of H2O2.
The UOx-induced hair coloring is effective and much milder than the direct oxidation
method using H2O2. UOx can function as a catalyst for p-phenylenediamine oxidation
as well as the H2O2 supplying source in the presence of UA to generate p-
benzoquinonediimine which is used for the preparation of hair-dyes (oxidative
polymerization of monomeric precursors) [219].
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
54
Table 2.3.1 Uricase based analytical methods
Sl.
No.
Analytical Method Reagent co-substrate Analyte determined Reference
01 Sp
ectr
opho
tom
eter
DHBS/4-AAP Uric acid (UA) in serum and urine [220]
Polyethyleneterephthalate/DHBS/4-AAP Serum urate [221]
Uricase/Catalase Uric acid [222]
Polyacrylonitrile/o-dianisidine/POD/UOx
/polyaniline/K2Cr2O7
UA in serum [223]
4-AAP/p-hydroxy)benzoicacid Serum UA [224]
N-methyl-N-(4-aminophenyl)-3-methoxy
aniline/TOOS/Ascorbate oxidase
UA in human serum [224]
p-hydroxybenzoate/4-AAP Serum UA assay [225]
Tetrazolium salt/CAT/FADH/1-methoxy-
5-methylphenazinium methyl sulfate
UA in serum [226]
Tribromophenol-4-AAP-POD UA in serum [227]
02 Molecular absorption
diode array
spectrometer
UOx-autotransducer molecular absorption
properties of HRP
UA in synthetic serum samples [228]
03 Chemometric tris(1,10-phenanthroline) /ferritin
Fe(phen)3]3+
AA, UA and DA in serum and
urine
[229]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
55
04 Flow injection analysis POD/ferrocene/CPE/Nafion/UOx/GOD H2O2 & D-glucose & UA in serum [230]
Poly(N,N-dimethylaniline) Detection of UA & Ascorbic acid [231]
05 Capillary
electrophoresis
Luminol/K3[Fe(CN)6] UA in urine and serum samples [232]
06 Chemiluminescence Pentacene/Peroxalate NPs/ UOx/alginate H2O2 and UA biosensor [233]
Microfluidic paper/Rhodanine UA biosensor [234]
07 Chemiluminescent
Biosensor
Diethylaminoethyl/Imidodiacetic acid/
GOD/GCE/luminol/Urate
Choline, glucose, glutamate,
lactate, lysine and UA
[235]
08 Infrared reflection
absorption spectroscopy
UOx onto Langmuir monolayers of stearic
acid/ Langmuir–Blodgett (LB) films
UA in Blood [236]
09 Bipotentiostatic Dodecylsulfate/poly(N-methylpyrrole) Detection of uric acid [237]
10
Am
pero
met
ric
Au NPs/MWCNT/carbodiimide linkage UA in serum [238]
Polystyrene/Polymaleimidostyrene UA biosensor [239]
Polyaniline UA in serum & real samples [240],
[241]
Amino acid nano composites UA biosensor [242]
Chitosan-poly(thiophene-3-boronic acid) UA in serum [243]
Gelatin/Glutaraldehyde/Teflon Urinary UA [244]
2-(2-mercaptoethylpyrazine)/4,4′-
dithiodibutyric acid
UA biosensor [245]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
56
11 Chronoamperometry SPCE/Cobalt phthalocyanine/cellulose
acetate/Polycarbonate membrane
Uric acid in urine [246]
3-aminopropyltriethoxysilane/Bis[sulfo
succinimidyl]suberate/indium-tin-oxide
UA biosensor [247]
12
Spectrofluorimeter UOx/Uric acid/Glycine/OH− buffer Uric acid in blood serum samples [248]
o-Phenylenediamine Serum UA assay [249]
Pyronine Y/Cu(II)/H2O2 UA in Urine [250]
Polyurethane-UOx-UA/Ru(dpp)3TMS2 UA in human blood serum [251]
Amplex red/silca sol-gel Uric acid in urine, serum & blood [252]
13 CV Poly(o-aminophenol) Uric acid in serum [253]
Theonine/SWCTs Endogenous & Physiological UA [254]
NiO/Titanium/Glass substrate UA biosensor [255]
Thionine-SWNTs UA in Cell lysate-serum samples [254]
Mercaptobenzimidazole/Ascorbic acid UA in human serum [256]
14 Coulometric Porous carbon felt electrode Uric acid in urine [257]
15 Conductometric Polyaniline-poly(n-butylmethacrylate)/Poly
(vinylmethylether)-poly(vinylethyl ether)
Urea and UA detection using
Urease and UOx
[258]
16 Reagentless biosensor UOx/ZnS Quantum Dots/L-cys biosensor UA biosensor [259]
17 96-well microtiter plate 8-azaxanthine, competitive inhibitor-UOx Urate oxidase activity rasburicase [260]
18 HPLC Cimetidine UA & creatinine in urine [261]
Semen samples diluted in Dithiothreitol/ AA and UA in human seminal [262]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
57
Ethanol-NaH2PO4 used as a mobile phase plasma
19 LC-MS m/z 167.0, corresponds to urate anion, and
m/z 169.0, corresponds to 1,3-15N2-UA
anion (isotope labeled UA as an internal
standard)/Trichloroacetic acid
Intracellular (Human umbilical
vein endothelial cells)/
Extracellular (plasma & urine) UA
[263]
20 Potentiometric assay ZnO/Au/Nafion UA in human blood serum [264]
21 Photo array sensor Metal oxide semiconductor/polymeric
enzyme biochip/UOx-POD
Serum UA [265]
22 Oxygen sensor UOx/Eggshell membrane/O2 electrode/UA UA in serum & urine [266]
23 Calorimetric biosensors UOx-Oxalate oxidase/biothermochips Kidney calculus indices [267]
24 Aqualytic O2 meter UOx-epoxy resin/polyamine cross linker Serum UA [268]
25 pH sensor Poly-N-isopropylacrylamide/Eu2Ti2O7 UA biosensor [269]
Sm2TiO5/Si substrate/UOx- alginate film Serum UA [270]
Note: CV: Cyclic voltammeter; DHBS: 3,5-Dichloro-2-hydroxybenzenesulfonic acid; 4-AAP: 4-aminophenazone; UOx: Uricase; UA:
Uric acid; FADH: Formaldehyde dehydrogenase; AA: Ascorbic acid; DA: Dopamine; CPE: Carbon paste electrode; GCE: Glassy carbon
electrode; SPCE: Screen printed carbon electrode; LC/MS- Liquid Chromatography-Tandem Mass spectrometry; TOOS: N-ethyl-N-
(hydroxy-3-sulfopropyl)-toluidine.
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
58
SECTION 2.4: CATALASE
2.4.1 Introduction
Catalase (H2O2 oxidoreductase, EC 1.11.1.6, CAT) comprises of four
ferriprotophorphyrin as prosthetic groups per molecule containing proteins that
include a variety of cytochromes, globins and peroxidases and is an important part of
body's antioxidant defenses, and is present in almost all living organisms. Acetobacter
peroxidans and Shigella dysenteriae [271] are exceptions and usually strict anaerobes
lack this enzyme. CAT is the typical marker enzyme of peroxisomes [272] and
participates in the degradation of H2O2 generated in the acyl-CoA oxidase reaction.
CAT defends against oxidative stress associated with pathologic conditions arising
out of cancer, diabetes, atherosclerosis, reperfusion injury, neurodegenerative disease
and aging [273].
In human tissues CAT plays a central role in controlling H2O2 concentration
by converting it into O2 and H2O [274], otherwise known as “catalatic” reaction. The
liver, erythrocytes and kidney are rich in CAT and more than 98 % of blood CAT is
derived from erythrocytes [275]. Other tissues such as brain, pancreas and serum have
CAT in very low concentration [276]. The normal range of CAT concentration in
healthy subjects is 120 U/mL ± 20 U/mL, whereas its concentration may vary
depending upon the condition of patients [277]. CAT, along with superoxide
dismutase (SOD) and glutathione peroxidase (GSH-Px), scavenges most of the levels
of O2-derived free radicals in mammalian cells and they function together as a
somatic oxidant defense [278]. Because ROS potently damages tissues, their final
conversion by SOD and CAT to harmless molecular O2 and H2O represents a
powerful antioxidative system, for preventing oxidative modifications of DNA,
proteins and lipids by blocking the chain reactions of free radicals produced in the
human body [279]. Free radicals are also responsible for many previously
unexplained diseases such as rheumatoid arthritis, Alzheimer’s, hyper-tension,
myocardial ishchemia, liver cells injury and carcinogenesis [280]. Hence,
measurement of CAT activity under certain conditions is valuable to assess the status
of vital defense system.
2.4.2 Structure of Catalase
Catalase is a tetramer of four polypeptide chains, each having over 500 amino
acids. It contains four porphyrin heme groups that allow the enzyme to react with
H2O2. The optimum pH of human CAT is approximately 7, and has a fairly broad
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
59
maximum. The pH optimum of other Catalases varies between 4 and 11 depending on
the species [281]. The optimum temperature also varies by species. The structure of
Catalase is shown in Figure 2.4.1.
Figure 2.4.1 Structure of Catalase (CAT)
2.4.3 General reaction pathway of CAT and its native iron states
HRP and CAT are the two enzymes which catalyze the decomposition of same
substrate: H2O2. However, the mechanisms of decomposition of H2O2 by these two
enzymes are completely different. HRP reduces H2O2 to H2O and CAT catalyzes the
breakdown of H2O2 into H2O and O2. In a ‘catalatic’ reaction of CAT enzyme, H2O2
oxidizes the heme iron of the resting enzyme to form an oxyferryl group with a π-
cationic porphyrin radical, termed compound I (Cpd I) as shown in Equation (1).
Where ‘Enz’ is the resting or ground state of the enzyme and Cpd refers to other states
of CAT.
The enzyme is first oxidized to a high-valent iron intermediate, known as Cpd
I which, in contrast to other hydroperoxidases, is reduced back to the resting state by
further reacting with H2O2. It is found that the Cpd I-H2O2 complex evolves to a Cpd
II-like species through the transfer of a hydrogen atom from the peroxide to oxoferryl
unit. The complete reaction sequence may involve two mechanisms that may be
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
60
operative: a His-mediated mechanism [282], which involves the distal His as an acid-
base catalyst mediating the transfer of a proton (associated with an electron transfer),
and a direct mechanism, in which one hydrogen atom transfer occurs [279]. Isotope
labeling kinetic studies on CAT have demonstrated that both the oxygen atoms of the
O2 molecule originate from the same H2O2 molecule [283]. Based on the crystal
structure of CAT, Fita and Rossmann (1985) proposed that the two hydrogens of
H2O2 are sequentially transferred to the oxoferryl unit of Cpd I, with the distal His
playing an active role in the reaction. Reaction 2 is a two-electron redox process and
clarifying whether it actually involves a two-electron transfer elementary step would
pose CAT in clear contrast with peroxidases, for which the resting state is restored in
two one-electron reduction processes [284].
Another state of CAT, Cpd II, results from the reduction of Cpd I by a single
electron as in Equation (3)
Recently, Rovira [285], provided evidence for the formation of a
hydroxyferryl form of Cpd II as in Equation (4)
Cpd II is formed during the catalatic reaction. It is inactive in the catalatic
reaction, but reverts spontaneously and relatively slowly to the active form (Enz).
Thus, during exposure to H2O2 that is generated at a constant rate, CAT can reach a
steady state in which much of it is inactive.
Mammalian CAT also has limited ability to act as a peroxidase. An example
of such a ‘peroxidatic’ reaction is the formation of acetaldehyde from ethanol and
H2O2. After the initiation of Cpd I in Equation (1), the reaction that takes place is
shown in Equation (5)
Peroxidatic activity is relatively slow. But in contrast, the catalatic rate of
mammalian CAT is highest among the known enzymatic rates. The catalatic rate is
simply proportional to H2O2 concentration over a wide range of H2O2 and it does not
follow the Michaelis–Menten kinetics [286]. Moreover, efforts made to determine the
reaction rates at high H2O2 concentrations are thwarted by the inactivation of CAT
that occurs at such levels. As a consequence, peroxidatic reactions are noticeable at
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
61
low H2O2 concentrations, but catalatic reaction predominates at higher H2O2
concentrations [287].
2.4.4 Physiological relevance of Catalase
CAT protects hemoglobin by removing more than half of the H2O2 generated
in normal human erythrocytes, which are exposed to substantial O2 concentrations
[288]. CAT activity has been observed in human placenta during early gestation
period [289]. Presence of L-DOPA, dopamine, hydroquinone and other
autooxidizable compounds in the fetal cells of rat inhibit the CAT activity resulting in
cell damage under mesencephalic cultures [290]. Studies on lymphocytes which is
activated by H2O2, stimulated in vitro by ROS to induce angiogenesis showed that
only enzyme CAT could block the activation [291]. Progesterone and various
synthetic steroids with progestin potencies counteract cell growth induced by H2O2,
through potent induction of CAT activities, in breast cancer cells and normal human
epithelial breast cells [292]. Increase in CAT activity has been observed in HIV
infected AIDS patients. Increases in serum CAT activity correlated with increase in
serum H2O2 scavenging ability and reached levels which decreased exogenous H2O2-
mediated injury to in vitro cultured endothelial cells without altering neutrophil
bactericidal activity or mononuclear cell cytotoxicity [293]. The toxic effects of ROS
are neutralized in the lens of eye by antioxidants such as ascorbic acid, vitamin E,
glutathione system (GSH peroxidase and reductase), SOD and CAT [294] and also in
the cataractous lenses of the type 2 diabetic and in senile group [295].
The decrease of CAT activity in malignant phenotype of mouse keratinocytes
studied u in-vitro model results in tumor progression suggesting that CAT should be
present in a sufficient amount to reduce the advancement of tumor growth [296].
Presence of GSH-Px, glutathione-S-transferase, CAT, Cu–Zn SOD activities and
malondialdehyde levels in erythrocytes of patients with non-small-cell lung cancer
(NSCLC) and small-cell lung cancer (SCLC), indicate significant changes in
antioxidant defense system in NSCLC and SCLC patients, which may lead to
enhanced action of oxygen radical [297].
A study of plasma thiobarbutyric acid-reacting substances (TBARS), blood
GSH (reduced glutathione) concentrations and erythrocyte antioxidant enzyme
activities (SOD/CAT/GSH-Px) in regularly menstruating women with ovulatory and
anovulatory menstrual cycles has made possible for the classification of subjects as
either ovulating or non-ovulating [298].
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
62
CAT has been implicated as an important factor in inflammation, mutagenesis,
prevention of apoptosis and stimulation of a wide spectrum of tumors [279]. It was
Fridovich [299], who suggested in 1975 that O2- and H2O2 interact to form the highly-
reactive hydroxyl radical according to the following equation
The reaction of oxidase systems like Hypoxanthine oxidase/xanthine results in
the formation of O2- which involves in the degradation breakage of single-stranded
DNA molecules and attacks on sugar moiety and the SOD and CAT enzymes protect
the DNA from these effects in presence of iron salts. Xanthine oxidase/xanthine
systems also render degradation and loss of viscosity in human synovial fluid system
wherein SOD and CAT act as antioxidants by protecting the biological system [300].
CAT has been reported for the prevention of target cells like nucleated cells,
which are readily killed by an enzymatically generated flux of superoxide. Addition
of human and murine erythrocytes blocks lethal damage to target cells and also
protects heterologous somatic cells against exogenous oxidant challenge [278]. Both
human SOD and CAT have been used in vivo as antioxidant therapy scavenging a
very small fraction of total oxidant production resulting decrease in lipid peroxidation
[301].
2.4.5 Applications of Catalase
The ability to degrade H2O2 has made CAT as one of the most industrially
significant enzymes. Immobilized CAT has useful applications in various industrial
fields including the removal of H2O2 used as oxidizing, bleaching or sterilizing agent
[302] and in the analytical field as a component of H2O2 or glucose biosensor
systems. Some important applications of Catalase are the following.
2.4.5.1 Catalase as biosensors
A brief introduction on principles of biosensors is provided in the second
chapter of Section 2.1: Peroxidase chapter, sub section 2.1.6.4. Using the catalatic
activity of CAT along with other oxidoreductase enzymes many types of biosensors
have been developed for the quantification of CAT activity, H2O2, and glucose. Some
of the recent Catalase based biosensors have been listed in Table 2.4.1.
2.4.5.2 Catalase in analysis of pollutants/pesticides in biological samples
Water pollutants such as pesticides, insecticides and heavy metals are the main
substances that were detected by amperometric biosensors via an enzyme alteration
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
63
process [303]. CAT is used for the determination of nitrite [304], cyanide [305], 3-
amino-1,2,4-triazole [306], [307] and azide [308] based on their inhibition effect on
enzyme activity in water and fruit juice samples.
2.4.5.3 Catalase in medical or clinical analysis
CAT, SOD and polyHb (poly hemoglobin) and a biodegradable polymeric
nano encapsule of Hb and enzymes (SOD-CAT, as lipid membrane artificial red
blood cells) have been used as blood substitutes [309]. PolyHb–SOD–CAT stabilize
the cross linked Hb resulting in decreased oxidative iron and heme release and also
reduce the formation of methemoglobin during the preparation of polyhemoglobin
[310]. Encapsulated CAT has been used successfully as an antioxidant against the
toxic effects of H2O2 in acatalesemic mice [308]. The first generation polyHb blood
substitutes (phase III clinical trials) have shown important clinical potential for certain
clinical conditions especially for short-term use in surgery [311].
CAT has also been used as a sperm motility extender in the cryopreservation
of bovine semen. Bovine sperm motility in egg yolk tris glycerol extender has been
protected by the use of pyruvate, metal chelators, bovine liver or oviductal fluid CAT
[312]. CAT can be used as a therapeutic agent in a variety of human diseases as CAT
can inhibit ROS-mediated tissue injury and tumor metastasis [313]. PEGylation-CAT
can effectively prevent the increase in the expression of epidermal growth factor
receptor metastatic tumor growth by detoxifying ROS as well as the peroxidation
[314]. CAT was encapsulated in biocompatible flexible non-ionic sugar esters (SEs)
nano-vesicles for topical administration in wound healing [315].
2.4.5.4 Catalase in food industry
In food industry, CAT has been used in the disposal of H2O2 prior to cheese
making [316]. CAT of cetyltrimethylammonium bromide-permeabilized cells was
effective in removing residual H2O2 from H2O2-treated fresh milk/heat pasteurized
milk [317]. In dairy industry, when H2O2 is used as a germicide for cold
pasteurization CAT is employed to remove the residual H2O2 present [318]. Other
important applications of CAT include desugaring of egg white [319], stabilization of
beverages by oxygen removal [320] and enzymic production of gluconic acid and
gluconic acid–fructose mixture from invert sugar [321].
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
64
Table 2.4.1 Catalase based analytical methods
Sl.No Analytical Method Reagent co-substrate Analyte determined Reference
01 S
pect
roph
otom
eter
Trinder’s reagent/Uric acid-Triton X-100 CAT in plasma, erythrocytes and liver [273]
Sodium cyanide CN contamination in aquatic biota [305]
Ammonium molybdate Serum CAT activity [322]
Ironporphyrin/3-amino-l,2,4-triazole CAT-hemolysates human, rat, mouse blood [323]
GOD/Aldehyde dehydrogenase CAT activity in erythrocytes [324]
K2Cr2O7/acetic acid CAT activity [325]
4-AAP/phenol/peroxidase CAT activity in soils [326]
p-nitrophenol p-nitrophenol inhibitory effect on CAT [327]
4-amino-3-hydrazino-5-mercapto-1,2,4-triazole CAT activity in small tissue samples [328]
o-Dianisidine CAT activity in honey samples [329]
3,3'-Diaminobenzidine-Glutaraldehyde Cytochemical detection of CAT [330]
NADP-NADPH Serum CAT activity [331]
NADPH-CAT Protection of CAT by NADPH [332]
Tetrahydrobiopterin (THB)-Ascorbate THB effect on ascorbate CAT [333]
Choline oxidase/4-aminopent-3-en-2-one Determination of Choline in liquor [334]
Oxalateoxidase/Aldehyde dehydrogenase Oxalate in serum and Urine samples [335]
Pyrocatechol-Isoniazid CAT activity-mycelia mats/culture media [336]
Methylhydroperoxide/Amino-1,2,4-triazole Formation of com I of CAT is investigated [337]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
65
02 FIA L-phenylalanine/L-amino acid oxidase L-phenylalanine in serum samples [338]
CNBr/Xanthine oxidase/Glyceraldehyde Monitoring inactivation-CAT by glycation [339]
03 Plate reader Ferrous ions/Thiocyanate Neural cell cultures of mesencephalon [340]
04 Amperometric ([Zn2CrABTS]LDH)/([Zn3AlCl]LDH)/NO2- Detection of nitrite in water samples [341]
AuNPs/MWCNTs/Chitosan CAT activity in liver homogenates of rats [342]
Polyacrylamide gel/Pt/Ag wire H2O2 biosensor [343]
05 Batch technique Glutaraldehyde/Chitosan/Cu(II) Metal sorption and CAT immobilization [344]
06 Calorimeter Uricase-CAT-tris buffer pH 9.0 Measurement of serum uric acid [345]
07 Chemiluminescence Luminol/Hypochlorite (NaOCl)/NaN3 Human erythrocytes and rat hepatocytes [346]
Dopamine/Luminol Study of auto oxidation of DA [347]
08 Conductometric HRP-CAT/Nitrite Detection of nitrite in water samples [304]
09
Cyc
lic v
olta
mm
eter
AuNPs/Graphene-NH2/GCE H2O2 biosensor [348]
Polyelectrolyte-encapsulated CAT/Au H2O2 biosensor [349]
MWCNTs/GCE H2O2 reagentless biosensor [350]
NiO/GCE H2O2 & nitrite reduction biosensor [351]
Cysteine/Si sol–gel/GCE/Au/Al3+ Neurotransmitter detection of CAT & Al3+ [352]
SWCNTs/Chitosan/GCE H2O2 biosensor and nitrite detection [353]
SOD-CAT/ Hg electrode Antioxidant properties of SOD and CAT [354]
10 Disk flotation
method
CAT CAT activity measurement [355]
11 ESR technique Sodium azide-CAT-H2O2 Detection of formation of Azidyl radical [356]
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
66
12 O2 Microsensor CAT/H2O2/degassed borate buffer CAT activity in green coffee sample [357]
13 Gasometric method H2O2/CAT Erythrocyte CAT activity [358]
14 HPLC Glutathione/o-phthalaldehyde derivative CAT activity in lysed human erythrocytes [359]
15 IMAC cryogel CibacronBlue/Poly(acrylamide-allyl glycidyl
ether) cryogel was chelated with Fe3+ ions
Effect of pH, protein conc., flow rate, and
temp. on ionic strength on CAT activity
[360]
16 ECIS 1-butyl-3-methylimidazoliumhexafluoro
phosphate/MWCNTs/GCE
Detection of ultra traces of H2O2 [361]
17 Oxygen sensor GOD-CAT/Pt-gas permeable membrane Glucose detection in blood [362]
18 pH sensitive
hydrogel
Sulfadimethoxine/GOD-CAT Glucose responsive hydrogel [363]
19 Batch plug-flow type Glutaraldehyde/6-aminohexanoicacid florisil Characterization and application [364]
20 Polarography Triphenylphosphine oxide SOD/CAT activity-human red blood cells [365]
21 Potentiometric HRP-CAT/H2O2/4-fluoro phenol CAT positive microorganisms [366]
22 Pyrolysis 2,2'-Azo-bis-(2-amidinopropane) (ABAP) Study of deactivation of SOD and CAT [367]
23 Radiometer Polypropylene/Nephrophan membrane H2O2 biosensor [368]
24 Raman spectra Isoniazid (INH)-CAT-peroxidase Activity of antituberculosis antibiotic INH [369]
25 Spectrofluorimeter Europium (III)–tetracycline Catalase activity [370]
26 Thermostat Gelatin/(CAT-Ca2+)/Glutaraldehyde Calcium in milk and water samples [371]
27 Titrimetric method KMnO4 CAT activity in waste water [372]
Note: 4-AAP: 4-aminoantipyrine; GCE: Glassy Carbon Electrode; Luminol: (5-amino-2,3-dihydro-1,4-phthalazinedione; IMAC:
Immobilized metal ion affinity chromatography cryogel; ECIS: Electrochemical Impedance Spectroscopy.
Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase
67
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